Stimulation of cell proliferation by hyaluronidase during in vitro aging of human skin fibroblasts

0531-5565/93 $6.00 + .00 Copyright(c) 1993 PergamonPress Ltd.

Experimental Gerontology, Vol. 28, pp. 59-68, 1993 Prinled in the USA. All rightsreserved.

S T I M U L A T I O N OF CELL PROLIFERATION BY H Y A L U R O N I D A S E D U R I N G IN VITRO AGING OF H U M A N SKIN FIBROBLASTS

MADELEINE MOCZAR a n d LADISLAS ROBERT Laboratoire de Biologie de Tissu ConjonctifCNRS URA 1460, Facult~ de Mfdecine, Universit~ Paris XII, 94010 Crfteil Cedex, France

Abstract - - The effect of the degradation of extracellular hyaluronan on the proliferation of human skin fibroblasts in serial cultures during in vitro aging was investigated. H u m a n skin fibroblasts at different time intervals from 3rd to 36th passages were exposed after plating to bovine testicular hyaluronidase. The enzyme treatment resulted in an increase in cell proliferation (cell number vs. time) as compared to the untreated control fibroblasts. The effect was dose dependent, reversible, and was independent of the type of the glycosidic linkage cleaved in hyaluronan. The increased proliferation was observed at all passages when untreated cells underwent mitosis. The degradation of hyaluronan induced cell proliferation up to the presenescent phase. Depletion of hyaluronan did not induce proliferation of postmitotic fibroblasts. The incorporation of 3Hglucosamine into hyaluronan decreased with increasing cell passages (increase of the number of population doublings). Twenty-fourth passage fibroblasts accumulated about two time less hyaluronan in the medium than ninth passage cultures. Following hyaluronidase treatment, the amount of newly synthesized, labeled hyaluronan increased in the medium. Accordingly, the fibroblasts restored the degraded hyaluronan even in the declining phase of proliferation (phase lII according to Hayrick). Key Words: hyaluronan, cell proliferation, glycosaminoglycans, flbroblasts, in vitro aging

INTRODUCTION HYALURONAN, A linear polysaccharide built up from glucuronyl-N-acetylglucosamine repeats (Rodrn 1980), is the major glycosaminoglycan component of the pericellular matrix synthesized by skin fibroblasts (Hedman et al., 1979). The configuration of its secondary structure is dependent on the water content of the environment (Heatly and Scott 1988). The tridimensional organization of hyaluronan can be represented by a hydrated random coil stabilized by hydrogen bonds (Laurent, 1970). Its physicochemical characteristics such as the large hydrated volume and viscoelasticity enable hyaluronan to regulate several biological functions, such as hydration and ion transport in the extracellular

Correspondence to: L. Robert. (Received 10 December 1991 ; Accepted 6 April 1992) 59

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M. MOCZAR AND L. ROBERI

space (Laurent and Fraser 1986). Its binding to specific cellular sites is involved in the modulation of cell proliferation and migration (Turley, 1989; Turley et al., 1990). The amount of hyaluronan in skin decreases sharply during maturation (Breen et al., 1976), as well as during in vitro cellular aging (Schachtschabel and Wever, 1978; Sluke et al., 1981). It decreases also in fibroblast cultures with increasing cell density (Hronowski and Anastassiades, 1980). Hyaluronan synthesis was shown to be required for the mitosis offibroblasts (Brecht et al., 1986); proliferating cells secrete higher amounts than quiescent cells (Matuoka et al., 1987). Hyaluronan biosynthesis by fibroblasts can be activated by several agents, such as hormones, cytokines, decreased pH, growth factors (Laurent and Fraser, 1986; Heldin et al., 1989), and by the enzymic degradation of the extracellular hyaluronan (Larnier et al., 1989). In this study, we investigated the effect ofhyaluronidase treatment on the proliferation of human skin fibroblasts during their progressive passages in a Hayflick type of in vitro cellular aging system (Hayflick and Moorhead, 1961 ; Hayflick, 1965: Hayflick, 1977). MATERIALS AND METHODS Dulbecco's essential medium and the reagents for cell cultures were obtained from Biochrom (Berlin). Fetal calf serum was purchased from Boehringer (Mannheim, Germany). Testicular hyaluronidase (EC 3.2.1.35) from bovine testes (295 U/mg), from Streptomyces type IX and type X (EC 3.2.1.36), and from leeches were obtained from Sigma (St. Louis, MO). Hyaluronidase from bovine testes (3813 U/mg) was purchased from Serva (Heidelberg, Germany), and 3H-glucosamine (8.4 Ci/mmol) from CEA (Saclay, France). Biogel P6DG desalting gel and columns were from Biorad (Richmond, CA). All reagents were of the highest purity commercially available. Cell culture

Fibroblasts were obtained from skin biopsies from 15- and 24-year-old healthy female donors after informed consent. The cells were cultured routinely in 75-cm ~ Falcon flasks in humidified 95% air/5% CO2 atmosphere in 5 ml Dulbecco's minimal essential medium (DMEM) containing 10% (v/v) fetal calf serum, 100 U/ml penicillin, 100 ug/ml streptomycin, and I ug/ml glutamine. Cell prol~li, ration assays

Cells were trypsinised in confluent cultures with 0.05% isotonic trypsin and 105 cells were seeded in Petri dishes (diameter 3.5 cm) containing 3 ml DMEM and supplements as described above. After 2 h the medium and the nonattached cells were aspirated off, and the cells were cultured in 3 ml fresh DMEM. 1. To investigate the proliferation as a function ofhyaluronidase concentration, 4th, 24th, and 36th passage cells were grown in 3 ml DMEM containing 0, 15, 75, and 150 U/ml testicular hyaluronidase (endo-B- l,~4-N-acetyl hexosaminidase). 2. Cell proliferation was studied at increasing numbers of passages in the presence of 75 U/ml testicular hyaluronidase. Medium was changed at the third and the fifth day of culture. Cell numbers were determined at the second, fifth, and seventh days following plating, by counting the cells released by trypsin. 3. To check a possible reversible effect of the hyaluronidase treatment, 21st passage cells

HYALURONIDASE AND CELL PROLIFERATION

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5. 6.

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were grown in the presence of 75 U/ml hyaluronidase for seven days, then the cells were trypsinised and further cultured in parallel passages in the absence and in the presence of hyaluronidase. To assay the effect of the type of glycosidic linkage cleaved, cells were cultured in the presence of 15 U/ml hyaluronidase from Streptomyces (endo-/3-N-acetyl- 1, ~ 4 glucosaminidase) and from leeches (endo-tS-1,~ 3-glucuronidase). Cultures were carried also in the presence of heat-inactivated testicular hyaluronidase, obtained after maintaining 150 U/ml hyaluronidase solution at 100°C for 3 min. Early passage cells were treated with 75 U/ml hyaluronidase after seeding as described in step 1. In parallel experiments the cells were plated, cultured, and at day 5 after plating were incubated with hyaluronidase for two days. In both experiments the cells were counted at seven days after seeding. (The experiments in steps 1-6 were carried out in triplicates.) The degradation ofhyaluronan was checked as described in Larnier et al., 1989. Briefly, hyaluronan was biosynthetically labeled with 3H-glucosamine as described below. Then cultures were incubated with hyaluronidase and the molecular size of labeled hyaluronan was determined in the medium in the treated and nontreated control cultures.

Biosynthetic labeling of hyaluronan The incubation of control and hyaluronidase-pretreated cultures with tritiated glucosamine was carried out as described (Larnier et al., 1989). Fibroblasts (l0 s cells/em 2) were treated with 75 U/ml testicular hyaluronidase for 3 h, and the medium was aspirated off. The residual enzyme was removed by repeated washings with phosphate-buffered saline from the cell layer. The cells were incubated with 20 uCi/ml 3H-glucosamine for 24 h. The medium was removed, the cells were washed two times with 1 ml PBS, and the medium and the rinse were pooled and stored at -20°C.

Characterization of 3H-hyaluronan Aliquot samples (up to 1 ml) from the culture medium were separated from the free radioactive precursor by gel chromatography on Biogel P6DG desalting columns ( 1 X 10 cm) equilibrated with 0.1 M sodium phosphate, 0.15 M sodium chloride, pH 5.3. The macromolecular label eluted in the void volume (5000 cpm) was assayed for its sensitivity to Streptomyces hyaluronidase (5 U/200 ul). Incubations with the enzyme were carried out at 37°C overnight. The obligosaccharides derived from hyaluronane and the nondegraded macromolecules were separated on Biogel P6 (1 X 40 cm) or on Biogel P6DG (1 X 10cm) columns eluted with phosphate-buffered saline, pH 7.2. The radioactivity of the eluates was measured, and the amount of hyaluronane was calculated from the amount of labeled oligosaccharides derived from hyaluronan recovered in the retarded volume of the column (Larnier et al., 1989).

Separation of 3H-hyaluronan Labeled macromolecules from the medium were separated from the 3H-glucosamine on Biogel P6DG ( 1 X 10 cm) columns equilibrated with 10 mM Tris HC1, pH 8.4. The macromolecular label eluted in the void volume was further separated by DEAE Trisacryl

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eluted with a linear 0-0.8 M NaCI gradient in 10 mM Tris HC1 (pH 8.4) as described (Brecht et al. 1986; Larnier et al., 1989). Gel permeation chromatography of 3H-hyaluronan was performed on Sepharose CL 4B (1 × 55 cm) eluted with 4 M guanidinium chloride, 0.01 M EDTA Na2, 0.05 M Tris, 0.05 M benzamidine HCI, pH 7.4. RESULTS Cell prol~feration

The fibroblasts were seeded and cultured in the absence and in the presence of increasing concentrations oftesticular hyaluronidase. The cell numbers at the fifth and seventh days following plating were higher in the treated cultures than in the control (nontreated) cultures. The cell numbers at the seventh day indicated that the effect increased with the amount of added enzyme and reached a plateau at about 75 U/ml hyaluronidase concentration (Fig. 1) The dose dependence was similar up to the 19th passage. Higher enzyme concentration (75 U/ml) was needed to increase the proliferation at later (24th) passages than in the early passages. To check the degradation of hyaluronan by the enzyme, the cultures containing biosynthetically labeled hyaluronan were exposed to hyaluronidase. In agreement with our previous data (Larnier et al., 1989), the analytical assays evidenced the cleavage of hyaluronan to oligosaccharides. In the treated cultures the time course indicated a sharp increase in cell proliferation between days 5 and 6 (Fig. 2). To confirm this observation by a different assay, early passage fibroblasts were exposed to the enzyme after plating and their proliferation was compared to that of cells incubated with the enzyme from day 5. At day 7, 6.61 ___0.13 × 105 cells were detected in the control (nontreated) cultures, whereas 7.91 _+ 0.10 × 105 and 7.12 _+ 0.49 × 105 fibroblasts were present in the cultures treated for 7 days and for the last 2 days of culture, respectively. 10 8 to •~

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FIG. 1. Dose-dependent stimulation o f proliferation o t h u m a n skin fibroblasts by testicular hyaluronidase. Fourth passage cells were plated, cultured, and the cells counted at the seventh day following plating as described in Materials and Methods. The results are m e a n s + SE. []4th, • -24th, and w-36th passage cells.

63

HYALURON1DASEAND CELL PROLIFERATION

8

7 'o

6

o

z

5

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2

I

I

I

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3

4

5

6

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7 Days

8

FIG. 2. T i m e course o f the proliferation offibroblasts in culture. Nineteenth passage fibroblasts were seeded, cultured, and the cells counted at the seventh day after seeding as described in Materials a n d Methods. D-control cells, I-cells in presence o f 7 5 U / m l bovine testicular hyaluronidase. Results are m e a n s _+ SE.

The action of the enzyme with progressing cell passages was assessed by counting the cells after 5 and 7 days of culture in the absence and in the presence of hyaluronidase. The cell numbers determined at the seventh day after plating in the control and treated cultures were plotted against the cell passage numbers on Fig. 3. As expected, the proliferation of fibroblasts (cell number vs. time) decreased with the progressing subcultivation or, in other terms, with the increase in population doublings. Up to the 24th passage hyaluronidase induced an about 1.4-fold increase in the cell proliferation. From the 3rd to the 14th passages, the results suggested about three and four population doublings per passage in the control and in the treated cultures respectively. The dose-dependent stimulation was detectable also from the 19th to 24th passages when the proliferation declined in the control cultures. In the late (36th) passage with a very low growth rate, hyaluronidase treatment (75 U/ml) hardly increased the proliferation by about 10%. It can be deduced that testicular hyaluronidase stimulated the proliferation of skin fibroblasts capable of mitosis up to the "presenescent" phase. The same extent of stimulation was obtained when testicular hyaluronidases 295 U/mg or 3813 U/mg were added at identical enzyme activities (75-100 U/ml) to the cultures. Accordingly, the effect was due to the enzyme activity and not to the contaminating proteins in the enzyme preparation. The stimulation of cell proliferation was lost when the cultures were exposed to heat-inactivated hyaluronidase. Hyaluronidase from Streptomyces (endo-¢3-1,4-N-acetyl glucosaminidase) and from leech (endo-13-1,3 glucuronidase) were also able to stimulate fibroblast proliferation (not shown). This shows that the observed effect was independent of the type of the glycosidic linkage cleaved.

Persistence of the effect When the fibroblasts were exposed to hyaluronidase continuously during two consecutive passages, the enzyme-pretreated cells proliferated at the same rate during the second

64

M. M O C Z A R A N D L. R O B E R T

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6

4 o

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10

20

30

Passage

40

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FIG. 3. Fibroblasts in the hyaluronidase-treated cultures and in the control cultures with progressingcell passages. Cells were cultured in the presence ofD 0 U/ml(controls) and of B 75 U/ ml bovine testicular hyaluronidase and counted at the seventh day after seeding as described in Materials and Methods. Results are means _+ SE.

t r e a t m e n t as the u n t r e a t e d c o n t r o l cells (Table 1). T h e s t i m u l a t i o n o f the proliferation was n o t additive. T o d e m o n s t r a t e the reversible effect o f h y a l u r o n i d a s e t r e a t m e n t , fibroblasts recovered f r o m the s t i m u l a t e d c u l t u r e s by t r y p s i n i z a t i o n were plated a n d c u l t u r e d further in the a b s e n c e o f h y a l u r o n i d a s e . After 7 days o f c u l t u r e n o significant difference was f o u n d b e t w e e n the p r o l i f e r a t i o n o f these cells a n d the u n t r e a t e d c o n t r o l cells. T h e s t i m u l a t i o n o f p r o l i f e r a t i o n i n the p r e v i o u s passage by h y a l u r o n i d a s e h a d n o effect o n the proliferation i n the s u c c e e d i n g passage w h e n the e n z y m e was a b s e n t ( T a b l e 1). It c a n be c o n c l u d e d that the s t i m u l a t i o n o f cell p r o l i f e r a t i o n b y h y a l u r o n i d a s e was reversible a n d necessitated the p r e s e n c e o f the e n z y m e .

TABLE

l . REVERSIBLE

STIMULATION

OF PROLIFERATION

HYALURONIDASE

OF HUMAN

SKIN FIBROBLASTS BY

IN C U L T U R E

C2"ll cuhure

Controls (22nd passage) Hyaluronidase treated* Hyaluronidase treated at two consecutive passages Hyaluronidase treated** following passage in absence of hyaluronidase Controls (23rd passage)

Cell No. X 1~ ~

1.86 + 0.26 2.64 _+ 0.36 2.56 + 0.27 1.76 _+ 0.10 1.68 + 0.20

Twenty-second passage cells were cultured for 7 days in the absence (control) and *in the presence of 75 U/ml testicular hyaluronidase. **Cellsat 22nd passage were treated with 75 U/ml hyaluronidase, trypsinized, and subcultured at the 23rd passage in the absence of the enzyme. Results are mean _+ SE.

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HYALURONIDASE AND CELL PROLIFERATION

Biosynthesis of hyaluronan To obtain information on the the biosynthetic response of fibroblasts to the enzymic degradation of hyaluronan with progressing subcultivation, the incorporation of tritiated glucosamine into hyaluronan was investigated at the 9th passage and, after the decrease of proliferative capacity, at the 24th passage. The incorporation oftritiated glucosamine into hyaluronan was lower in the 24th passage cultures than in the 9th passage cultures. After pretreatment of the cultures with hyaluronidase the amount of newly synthesized, labeled hyaluronan increased in the medium at both passages (Fig. 4). This result is in agreement with our previous experimental data on the stimulation of hyaluronan biosynthesis by hyaluronidase-treated fibroblasts (Larnier et al., 1989). The amount of labeled hyaluronan produced by the hyaluronidase-pretreated 24th passage cells equaled the amount released into the medium by the untreated early passage cells. The hyaluronan from hyaluronidasepretreated cultures was mainly of low hydrodynamic size. It was eluted as a broad included peak on Sepharose CL4B chromatography in 4 M guanidinium chloride. Although hyaluronan synthesis was stimulated at this late passage, this stimulation did not restore the decreased hyaluronan biosynthesis after the decline of cell proliferation. DISCUSSION Fibroblast cultures with limited life span were frequently used as in vitro models for the study of biological aging of cells (Hayrick and Moorhead, 1961; Hayrick, 1965; Hayrick, 1977; Macieira-Coelho, 1973) The interactions of cells with extracellular matrix components such as proteoglycans and glycosaminoglycans were recognized as regulatory events of cell behavior (Kjellrn and Lindahl, 1991). High concentration of exogenous hyalu-

31

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FIG. 4. Accumulation of 3H-hyaluronan in the culture medium of fibroblasts. Cells were incubated with 3H-glucosamine and the labeled hyaluronan characterized as described in Materials and Methods. W-control cells, D-cells pretreated with bovine testicular hyaluronidase. Results are means _+ SE.

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ronan ( > 500 ~g/ml) could slightly inhibit the proliferation of human flbroblasts (WI-38) in short- or long-term cultures. It did not modify, however, the in vitro aging in terms of population doublings (Wever et al., 1980). The aim of our investigations was to obtain information on the potential role of endogenous hyaluronan, especially of its macromolecular nature, in the in vitro aging of skin fibroblasts. Extracellular hyaluronan can be readily degraded in fibroblast cultures by hyaluronidases. In our previous studies we evidenced a transient stimulation ofhyaluronan synthesis following hyaluronidase treatment of early passage fibroblasts (Larnier et al., 1989). This stimulation requires the degradation of cell-associated hyaluronan, is specific, and was not inhibited by the oligosaccharides from hyaluronan. The enzymic depletion adopted in our present model revealed the sensitivity of skin fibroblasts to the molecular mass of the endogenous hyaluronan. The most marked effect of hyaluronan degradation on cell proliferation was observed between the fifth and seventh days of culture. This result suggests that fragmentation of extracellular hyaluronan is not the only factor involved in this phenomenon. To obtain information on the possible role of the cell density, fibroblasts 5 days after seeding (approximately 50 X 103cells/cm 2) were incubated with the enzyme for 2 more days. The stimulation of proliferation in this condition seems to be consistent with the above contention. It can be noted that hyaluronan depletion affects proliferation at cell densities where hyaluronan synthesis was reported to decrease sharply in rat skin fibroblasts (Hronowski and Anastassiades, 1980). One plausible interpretation of the above observations would be the "sensing" of the loss of hyaluronan by specific cell-binding sites (Turley, 1989; Turley et al., 1990) and the age-dependent variation of these interaction sites. These events may be well involved in the signaling of cell proliferation. The low-molecular-size oligosaccharides from hyaluronan did not associate with the fibroblasts (Larnier et al., 1989); thus, we may argue that such fragments are not involved in the stimulation of cell proliferation. Although the hyaluronidase-treated cultures could reach higher cell densities than the control cultures, the stimulating effect could not counterbalance the decreased proliferation during senescence in Phase III cultures. This decline appears to reflect a loss of the efficiency of the signaling mechanism in Phase II1 (Hayrick, 1977). We attempted to obtain evidence on the capacity of cells to restore the degraded hyaluronan during in vitro aging. The decrease in the incorporation of tritiated glucosamine indicated a lower hyaluronan synthesis after the decline of cell proliferation as compared to the early passage cultures. Nevertheless 24th passage cells synthesized higher amounts ofhyaluronan after enzyme treatment than the untreated controls. This effect on the synthesis shows that fibroblasts can restore depleted hyaluronan even when their capacity to proliferate is decreasing. The apparent parallelism between resynthesis ofhyaluronan and cell proliferation is supported by the increased hyaluronan synthesis by proliferating cells (Matuoka el al., 1987). On the other hand, we should consider that the different stimulation ofhyaluronan synthesis by growth factors did not correlate with their mitogenic activities (Heldin el al., 1989). In this connection, the observed changes in cell proliferation can not be attributed to a single mechanism. The UDP sugars required for the biosynthesis of hyaluronan and of sulfated glycosaminoglycans are derived from different cellular pools (von Figura et al., 1973). It can be assumed that the decreased hyaluronan synthesis is a specific phenomenon during in vitro aging and cannot be related to the slowdown of sulfated glycosaminoglycan synthesis in the senescent fibroblast cultures (Schachtschabel and Wever, 1978; Sluke et al., 1981;

HYALURONIDASE AND CELL PROLIFERATION

67

Vogel et al., 1981) The elucidation of the nature of the interactions between fibroblasts and hyaluronan as well as the mechanisms of signaling implicated in the observed stimulation of cell proliferation require further studies. Evidence is provided, however, that the amount and/or molecular size ofpericellular hyaluronan can influence fibroblast proliferation in a passage-dependent matter. Acknowledgments - - This work was supported by CNRS (URA 1460), by grant from INSERM(CRAMIF), and by the Scientific Council of the Medical Faculty of University Paris XII. We thank Mrs. A. Duchesnay for skillful technical assistance.

REFERENCES BRECHT, M., MAYER, V., SCHLOSSER, E. & PREM, P. Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem Z 239, 445-450, 1986. BREEN, M., WEINSTE1N, H.G., BLACIK, L.J., BORCHERDING, M.S., and SITT1G, R.A. Microanalysis and characterization ofglycosaminoglycans from human tissue via zone electrophoresis. In: Methods in Carbohydrate Chemistry, Whistler, R.L. and BeMiller, J.N. (Editors), pp. 101-115, Academic Press, New York, NY, 1976. HAYFLICK, L. The limited in vitro life time of human diploid cell stains. Exp. CellRes. 37, 614-636, 1965. HAYFLICK, L, The cellular basis of biological aging. In: Handbook of the Biology of Aging, Finch, C.E. and Hayflick, L. (Editors), pp. 159-186, Van Nostrand-Reinhold, New York, NY, 1977. HAYFLICK, L. and MOORHEAD, P.S. The serial cultivation of human diploid cell stains. Exp. Cell Res. 25, 585-621, 1961. HEATLY, F. and SCOTT, J.E. A water molecule participates in the secondary structure ofhyaluronan, Biochem. J~ 254, 489-493, 1988. HEDMAN, K., KURKINEN, M., ALITALO, K., VAHERI, A., JOHANSSON, S., and HOOK, M. Isolation of the pericellular matrix of human fibroblast cultures. J. Cell Biol. 81, 83-91, 1979. HELDIN, P., LAURENT, T.C., and HELDIN, C.H. Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts. Biochem. Z 258, 919-1022, 1989. HRONOWSKI, L. and ANASTASSIADES, T.P. The effect of cell density on net rates of glycosaminoglycan synthesis and secretion by cultured fibroblasts. J. Biol. Chem. 255, 10091-10099, 1980. KJELLEN, L. and LINDAHL, U. Proteoglycans: Structures and interactions. Annu. Rev. Biochem. 60, 443-475, 1991. LARNIER, C., KERNEUR, C., ROBER, L., and MOCZAR, M. Effect of testicular hyaluronidase on hyaluronate synthesis by human skin fibroblasts in culture. Biochim. Biophys. Acta 1014, 145-152, 1989. LA U RENT, T.C. Structure of hyaluronic acid. In: Chemistry and Molecular Biology of the Intercellular Matrix. Vol. 2, Balazs, E.A. (Editor), pp. 703-732, Academic Press, London, 1970. LAURENT, T.C. and FRASER, J.R.E. The properties and turnover of hyaluronan. Ciba Found. Syrup. 124, 823, 1986. MACIE1RA-COELHO, A. Aging of connective tissues. In: Frontiers of Matrix Biology. Vol. 1. Skin, Robert, L. (Editor), pp. 46-77, Karger, Basel, Switzerland, 1973. MATUOKA, K., NAMBA, M., and MITSUI, Y. Hyaluronate synthase inhibition by normal and transformed fibroblasts during growth reduction. J. Cell Biol. 104, 1105-1115, 1987. RODI~N, L. Structure and metabolism of connective tissue proteoglycans. In: Biochemistry tfGlycoproteins and Proteoglycans. Lennarz, W.J. (Editor), pp.261-371, Plenum Press, New York, NY, 1980. SCHACHTSCHABEL, D.O. and WEVER, J. Age related decline in the synthesis ofglycosaminoglycans by cultured human fibroblasts (WI 38). Mech. Ageing Dev. 8, 257-264, 1978. SLUKE, G., SCHACHTSCHABEL, D.O., and WEVER, J. Age related changes in the distribution pattern of glycosaminoglycans synthesized by cultured human diploid fibroblasts (WI 38). Mech. Ageing Dev. 16, 1927, 1981. TURLEY, E.A. Hyaluronic acid stimulates protein kinase activity in intact cells and in isolated protein complex. J. Biol. Chem. 264, 8951-8955, 1989. TURLEY, E.A., BRASSEL, P. and MOORE, D. A hyaluronan-binding protein shows a partial and temporally regulated codistribution with actin on locomoting chick heart fibroblasts. Exp. CellRes. 187, 243-249, 1990.

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VOGEL, K.G., KENDALL, V.F., and SAPIEN, R.E. Glycosaminoglycan synthesis and composition in human fibroblasts during in vitro cellular aging (IMR 90). J. Cell. Physiol. 107, 271-281, 1981. VON FIGURA, K., KIOKOWSKI, W., and BUDDECKE, E. Differently labelled glucosamine precursor pools for the biosynthesis of hyaluronate and heparan sulfate. Eur. J. Biochem. 40, 89-94, 1973. WEVER, J., SCHATCHTSCHABEL, D.O., SLUKE, G., and WEVER, G. Effect of short and long term treatment with exogenous glycosaminoglycans on growth and glycosaminoglycan synthesis of human fibroblasts (WI 38) in culture. Mech. Ageing Dev. 14, 89-99, 1980.